Apparatus for using optical tweezers to manipulate materials

ABSTRACT

A method and apparatus for control of optical trap arrays and formation of particle arrays using light that is in the visible portion of the spectrum. The method and apparatus provides a laser and a time variable diffractive optical element to allow dynamic control of optical trap arrays and consequent control of particle arrays and also the ability to manipulate singular objects using a plurality of optical traps. By avoiding wavelengths associated with strong absorption in the underlying material, creating optical traps with a continuous-wave laser, optimizing the efficiency of individual traps, and trapping extended samples at multiple points, the rate of deleterious nonlinear optical processes can be minimized.

[0001] This invention was made with U.S. Government support underContract No. DMR-9730189 awarded by the National Science Foundation,through the MRSEC Program of the National Science Foundation under AwardNo. DMR-9808595, and through a GAANN fellowship from the Department ofEducation. The U.S. Government also has certain rights to the invention.

[0002] The present invention is directed generally to a method andapparatus for control of optical traps or optical tweezers. Moreparticularly, the invention is directed to optical tweezers formed usingvisible light that can be used to manipulate a variety of lightsensitive materials, such as living biological materials, withoutsubstantial damage or deleterious effects upon the material beinginvestigated or manipulated.

[0003] It is known to construct optical tweezers using optical gradientforces from a single beam of light to manipulate the position of a smalldielectric particle immersed in a fluid medium whose refractive index issmaller than that of the particle. The optical tweezer technique hasbeen generalized to enable manipulation of reflecting, absorbing and lowdielectric constant particles as well.

[0004] Some systems have therefore been developed which can manipulate asingle particle by using a single beam of light to generate a singleoptical trap. To manipulate multiple particles with such systems,multiple beams of light must be employed. The difficulty of creatingextended multiple-beam traps using conventional optical tweezermethodology inhibits their use in many potential commercial applicationssuch as the inspection of biological materials generally, and also thefabrication and manipulation of nanocomposite materials includingelectronic, photonic and opto-electronic devices, chemical sensor arraysfor use in chemical and biological assays, and holographic and computerstorage matrices.

[0005] An optical tweezer uses forces exerted by an intense and tightlyfocused beam of light to trap and manipulate dielectric particles,typically in fluid media. Prior descriptions of optical tweezersemphasized their potential utility for biological applications such ascapturing cells, or their components, for research, diagnosticevaluation, and even therapeutic purposes. These same reports alsoemphasized the inherent and persistent occurrence of damage or changescaused by optical trapping methods when using visible light. Inparticular, it has been observed that green light of wavelength λ=514.5nm from an Ar ion laser has caused various deleterious effects onbiological material: red blood cells literally explode, the chloroplastsof green plant cells were destroyed, and the continued application of agreen laser light has caused the death of trapped ciliated bacteria.Damage from the green laser light in the first two examples clearlyresulted from strong absorption of green light by hemoglobin andchlorophyll, respectively, leading to rapid heating and catastrophicdestruction. The mechanism of the third type of damage, dubbed“opticution” by those in the art, was not immediately obvious.Subsequent studies have identified optically-induced mutagensis to be alikely mechanism for the cells' death by virtue of the use of opticaltweezers.

[0006] Considerably less damage to biological materials was observedwhen comparable materials were optically trapped with infrared lightfrom a Nd:YAG laser operating at λ=1064 nm. Largely on the basis ofthese and similar early observations with a single optical tweezer,researchers came to the conclusion that infrared illumination isoperationally superior to visible illumination for optically trappingbiological materials. That is, use of infrared light did not cause anyapparent deleterious effect upon biological material.

[0007] Laser-induced damage can be desirable, however, in specialcircumstances. For example, pulsed optical tweezers operating at λ=532nm have been singled out for their ability to cut biological materials,such as chromosomes. Optical tweezers used in this way are known asoptical scissors or optical scalpels. Even so, the prospects fornondestructively trapping biological materials with visible light hadpreviously been considered by those in the art to be an unacceptablemethod of optical trapping and manipulation due to the well documentedand accepted deleterious effect on biological material.

[0008] It is therefore an object of the invention to provide an improvedmethod and system for using at least one optical trap from light in thevisible or ultraviolet portion of the spectrum.

[0009] It is also an object of the invention to provide a novel methodand apparatus for control of visible light optical traps that has anincreased level of efficiency, effectiveness and safety for use.

[0010] It is yet another object of the invention to provide a novelmethod and apparatus for control of visible light optical traps that isrelatively simple to align by virtue of using light visible to the humaneye.

[0011] It is still a further object of the invention to provide a novelmethod and apparatus for control of visible light optical traps wherelocalized regions can be accurately trapped.

[0012] It is an additional object of the invention to provide a novelmethod and apparatus for control of visible light optical traps whereinthe samples being manipulated are not overly heated or otherwise altereddue to light absorption.

[0013] It is yet a further object of the invention to provide a novelmethod and apparatus for control of optical traps wherein the opticaltraps have highly improved tracking accuracy.

[0014] It is an another object of the invention to provide a novelmethod and apparatus for using visible light for optical tweezers foruse on any material whose electronic, mechanical, chemical or biologicalstate is highly sensitive to optical tweezers having a high intensitylight pattern.

[0015] It is still another object of the invention to provide a novelmethod and apparatus for control of optical traps with a variable powerlevel which provides efficient optical trapping but without alterationof the desired chemical, biological, electronic or mechanical state ofthe material.

[0016] In accordance with the above objects, it has been discovered thatdamage or unwanted alterations inflicted by visible optical tweezers onbiological matter, and other materials sensitive to high intensitylight, can be reduced to acceptable, de minimis or even zero dimensionsand levels, in part through appropriate design of the optical trappingsystem and method. In addition, it is believed that wavelengths in theultraviolet can also be used for particular small size objects andparticular types of materials by taking advantage of the features ofthis invention. Consequently, such optical tweezers can have widespreadapplications in biological systems and other systems having lightsensitive materials and possess a number of advantages over infraredoptical tweezers.

[0017] Other objects, features and advantages of the present inventionwill be readily apparent from the following description of the preferredembodiments thereof, taken in conjunction with the accompanying drawingsdescribed below wherein like elements have like numerals throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 illustrates a method and system which includes someconventional features for a single optical tweezer;

[0019]FIG. 2 illustrates a method and system which includes someconventional features for a single, steerable optical tweezer;

[0020]FIG. 3 illustrates a method and system using a diffractive opticalelement;

[0021]FIG. 4 illustrates another method and system using a tiltedoptical element relative to an input light beam;

[0022]FIG. 5 illustrates a continuously translatable optical tweezer(trap) array using a diffractive optical element;

[0023]FIG. 6 illustrates a method and system for manipulating particlesusing an optical tweezer array while also forming an image for viewingthe optical trap array;

[0024]FIG. 7A illustrates an image of a four by four array of opticaltweezers (traps) using the optical system of FIG. 6; and FIG. 7Billustrates an image of one micrometer diameter silica spheres suspendedin water by the optical tweezers of FIG. 7A immediately after thetrapping illumination has been extinguished, but before the spheres havediffused away;

[0025]FIG. 8 illustrates a method and system for manipulating particlesusing an optical tweezer array while also accelerating the filling ofoptical traps;

[0026] FIGS. 9A-9D illustrate the use of optical trap controlmethodology wherein the optical traps are formed by a holographicdiffractive optical element; and

[0027]FIG. 10 illustrates the use of optical trap control methodologywherein a microscope images the particles and a personal computer isused to identify the particles and calculate a phase only hologram totrap the particles.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In order to best understand the improvement of the invention,FIGS. 1 and 2 illustrate several methods and systems which include someconventional features. In an optical tweezer system 10 of FIG. 1,optical gradient forces arise from use of a single beam of light 12 tocontrollably manipulate a small dielectric particle 14 dispersed in amedium 16 whose index of refraction, n_(m), is smaller than that of theparticle 14. The fundamental nature of the optical gradient forces iswell known, and also it is understood that the principle has beengeneralized to allow manipulation of reflecting, absorbing and lowdielectric constant particles as well. Any of these techniques can beimplemented in the context of the invention improvements describedhereinafter and will be encompassed by use of the terminology opticaltweezer, optical trap and optical gradient force trap hereinafter.

[0029] The optical tweezer system 10 is applied by using a light beam 12(such as a laser beam or other very high intensity light sources)capable of applying the necessary forces needed to carry out the opticaltrapping effect needed to manipulate a particle. The objective of aconventional form of the optical tweezer 10 is to project one or moreshaped beams of light into the center of a back aperture 24 of aconverging optical element (such as an objective lens 20). As noted inFIG. 1 the light beam 12 has a width “w” and having an input angle Ørelative to an optical axis 22. The light beam 12 is input to a backaperture 24 of the objective lens 20 and output from a front aperture 26substantially converging to a focal point 28 in focal plane 30 ofimaging volume 32 with the focal point 28 coinciding with an opticaltrap 33. In general, any focusing optical system can form the basis forthe optical tweezer system 10.

[0030] In the case of the light beam 12 being a collimated laser beamand having its axis coincident with the optical axis 22, the light beam12 enters the back aperture 24 of the objective lens 20 and is broughtto a focus in the imaging volume 32 at the center point c of theobjective lens focal plane 30. When the axis of the light beam 12 isdisplaced by the angle Ø with respect to the optical axis 22, beam axis31 and the optical axis 22 coincide at the center point B of the backaperture 12. This displacement enables translation of the optical trapacross the field of view by an amount that depends on the angularmagnification of the objective lens 20. The two variables, angulardisplacement Ø and varying convergence of the light beam 12, can be usedto form the optical trap at selected positions within the imaging volume32. A multiple number of the optical traps 33 can be arranged indifferent locations provided that multiple beams of light 12 are appliedto the back aperture 24 at the different angles Ø and with differingdegrees of collimation.

[0031] In order to carry out optical trapping in three dimensions,optical gradient forces created on the particle to be trapped mustexceed other radiation pressures arising from light scattering andabsorption. In general this necessitates having the wave front of thelight beam 12 to have an appropriate shape at the back aperture 24. Forexample, for a Gaussian TEM_(∞) input laser beam, the beam diameter wshould substantially coincide with the diameter of the back aperture 24.For more general beam profiles (such as Gauss-Laguerre) comparableconditions can be formulated.

[0032] In another system in FIG. 2 which includes some conventionalfeatures, the optical tweezer system 10 can translate the optical trap33 across the field of view of the objective lens 20. A telescope 34 isconstructed of lenses L1 and L2 which establishes a point A which isoptically conjugate to the center point B in the prior art system ofFIG. 1. In the system of FIG. 2 the light beam 12 passing through thepoint A also passes through the point B and thus meets the basicrequirements for performing as the optical tweezer system 10. The degreeof collimation is preserved by positioning the lenses L1 and L2 as shownin FIG. 2 to optimize the transfer properties of the telescope 34. Inaddition, the magnification of the telescope 34 can be chosen tooptimize angular displacement of the light beam 12 and its width w inthe plane of the back aperture 24 of the objective lens 20. As statedhereinbefore, in general several of the light beams 12 can be used toform several associated optical traps. Such multiple beams 12 can becreated from multiple independent input beams or from a single beammanipulated by conventional reflective and/or refractive opticalelements.

[0033] In one preferred embodiment of an overall optical manipulationsystem shown in FIG. 3, arbitrary arrays of optical traps can be formed.A diffractive optical element 40 is disposed substantially in a plane 42conjugate to back aperture 24 of the objective lens 20. Note that only asingle diffracted output beam 44 is shown for clarity, but it should beunderstood that a plurality of such beams 44 can be created by thediffractive optical element 40. The input light beam 12 incident on thediffractive optical element 40 is split into a pattern of the outputbeam 44 characteristic of the nature of the diffractive optical element40, each of which emanates from the point A. Thus the output beams 44also pass through the point B as a consequence of the downstream opticalelements described hereinbefore.

[0034] The diffractive optical element 40 of FIG. 3 is shown as beingnormal to the input light beam 12, but many other arrangements arepossible. For example, in FIG. 4 the light beam 12 arrives at an obliqueangle β relative to the optic axis 22 and not at a normal to thediffractive optical element 40. In this embodiment, the diffracted beams44 emanating from point A will form optical traps 50 in focal plane 52of the imaging volume 32 (seen best in FIG. 1). In this arrangement ofthe optical tweezer system 10 an undiffracted portion 54 of the inputlight beam 12 can be removed from the optical tweezer system 10. Thisconfiguration thus enables processing less background light and improvesefficiency and effectiveness of forming optical traps.

[0035] The diffractive optical element 40 can include computer generatedholograms which split the input light beam 12 into a preselected desiredpattern. Combining such holograms with the remainder of the opticalelements in FIGS. 3 and 4 enables creation of arbitrary arrays in whichthe diffractive optical element 40 is used to shape the wavefront ofeach diffracted beam independently. Therefore, the optical traps 50 canbe disposed not only in the focal plane 52 of the objective lens 20, butalso out of the focal plane 52 to form a three-dimensional arrangementof the optical traps 50.

[0036] In the optical tweezer system 10 of FIGS. 3 and 4, also includedis a focusing optical element, such as the objective lens 20 (or otherlike functionally equivalent optical device, such as a Fresnel lens) toconverge the diffracted beam 44 to form the optical traps 50. Further,the telescope 34, or other equivalent transfer optics, creates a point Aconjugate to the center point B of the previous back aperture 24. Thediffractive optical element 40 is placed in a plane containing point A.

[0037] In another embodiment, arbitrary arrays of the optical traps 50can be created without use of the telescope 34. In such an embodimentthe diffractive optical element 40 can be placed directly in the planecontaining point B.

[0038] In the optical tweezer system 10 either static or time dependentdiffractive optical elements 40 can be used. For a dynamic, or timedependent version, one can create time changing arrays of the opticaltraps 50 which can be part of a system utilizing such a feature. Inaddition, these dynamic optical elements 40 can be used to actively moveparticles and matrix media relative to one another. For example, thediffractive optical element 40 can be a liquid crystal phase arrayundergoing changes imprinted with computer-generated holographicpatterns.

[0039] In another embodiment illustrated in FIG. 5, a system can beconstructed to carry out continuous translation of the optical tweezertrap 50. A gimbal mounted mirror 60 is placed with its center ofrotation at point A. The light beam 12 is incident on the surface of themirror 60 and has its axis passing through point A and will be projectedto the back aperture 24. Tilting of the mirror 60 causes a change of theangle of incidence of the light beam 12 relative to the mirror 60, andthis feature can be used to translate the resulting optical trap 50. Asecond telescope 62 is formed from lenses L3 and L4 which creates apoint A′ which is conjugate to point A. The diffractive optical element40 placed at point A′ now creates a pattern of diffracted beams 64, eachof which passes through point A to form one of the tweezer traps 50 inan array of the optical tweezers system 10.

[0040] In operation of the embodiment of FIG. 5, the mirror 60translates the entire tweezer array as a unit. This methodology isuseful for precisely aligning the optical tweezer array with astationary substrate to dynamically stiffen the optical trap 50 throughsmall-amplitude rapid oscillatory displacements, as well as for anyapplication requiring a general translation capability.

[0041] The array of the optical traps 50 also can be translatedvertically relative to the sample stage (not shown) by moving the samplestage or by adjusting the telescope 34. In addition, the optical tweezerarray can also be translated laterally relative to the sample by movingthe sample stage. This feature would be particularly useful for largescale movement beyond the range of the objective lens field of view.

[0042] In another embodiment shown in FIG. 6 the optical system isarranged to permit viewing images of particles trapped by the opticaltweezers 10. A dichroic beamsplitter 70, or other equivalent opticalbeamsplitter, is inserted between the objective lens 20 and the opticaltrain of the optical tweezer system 10. In the illustrated embodimentthe beamsplitter 70 selectively reflects the wavelength of light used toform the optical tweezer array and transmits other wavelengths. Thus,the light beam 12 used to form the optical traps 50 is transmitted tothe back aperture 24 with high efficiency while light beam 66 used toform images can pass through to imaging optics (not shown).

[0043] An illustration of one application of an optical system is shownin FIGS. 7A and 7B. The diffractive optical element 40 is designed tointeract with the single light beam 12 to create a 4×4 array ofcollimated beams. A 100 mW frequency doubled diode-pumped Nd:YAG laseroperating at 532 nm provides a Gaussian TEM_(∞) form for the light beam12. In FIG. 7A the field of view is illuminated in part by laser lightbackscattered by sixteen silica spheres trapped in the array's sixteenprimary optical tweezers 10. The 1 μm diameter spheres are dispersed inwater and placed in a sample volume between a glass microscope slide anda 170 μm thick glass coverslip. The tweezer array is projected upwardthrough the coverslip and is positioned in a plane 8 μm above thecoverslip and more than 20 μm below the upper microscope slide. Thesilica spheres are stably trapped in three-dimensions in each of thesixteen optical tweezers 10.

[0044] In FIG. 7B is shown the optically-organized arrangement ofspheres {fraction (1/30)} second after the optical tweezers 10 (traps)were extinguished but before the spheres had time to diffuse away fromthe trap site.

Adaptive Tweezer Mode

[0045] In various embodiments the basic optical trap modes describedhereinbefore can be used in various useful methodologies. Furthermore,other embodiments include apparati and systems which can be constructedto apply these methods to enhance operation and use of the opticaltraps. In particular, the optical traps can be controlled and modified,and various embodiments employing these features are describedhereinafter.

[0046] A variety of new uses and applications of optical traps can arisefrom time varying construction and dynamic change of optical trapconfiguration. In one form of the invention an array of optical trapscan be advantageously manipulated in the manner shown in FIG. 8. In theillustrated optical system 100, the diffractive optical element 102splits the collimated laser beam 104 into several (two or more) laserbeams 106 and 108. Each of the several laser beams 106 and 108 aretransferred into a separate optical trap in an object plane 118. Each ofthese several laser beams 106, 108 are transferred to the back aperture110 of the objective beam 112 by action of a conventional opticalarrangement, such as the telescope formed by the laser 114 and 116. Theobjective lens 112 focuses each of these several beams 106, 108. In apreferred form of the invention a movable knife edge 120 is disposed tobe movable into the path of the several laser beams 106, 108, therebyenabling selective blocking of any selected one(s) of the several laserbeams to selectively prevent formation of a portion of the opticaltraps. Such a methodology and structure enables construction of anydesired array of optical traps by use of appropriately designed knifeedges or apertured knife edge structure and like such structures.

[0047] An illustration of the use of such optical trap controlmethodology is shown in FIG. 9 wherein optical traps are formed by aholographic form of diffractive optical element 122. The movable knifeedge 120 of FIG. 8 can block all but one line 124 of its optical traps,and by systematically moving the knife edge 120, each of the lines 124can be established. This enables systematic filling of optical traps 132with particles 126. This methodology allows filling of the optical traps132 with a variety of different types of the particles 126 and alsoavoids the typical problem of the particles 126 tending to fillpreferentially the outer portions of an array of optical traps. Suchpreferential filling can block filling of the inner optical traps. Thiscontrolled formation of the optical traps also permits precisionformation and change of optical trap arrangements.

[0048] In addition to exerting detailed control over filling of an arrayof the optical traps 132, devices can be provided to accelerate fillingof the optical traps. For example, in FIG. 8 is shown a functional block128 indicative of a device to (1) output selected particles 126 (seeFIG. 10), (2) apply the particles 126 under pressure differential(through electrophoresis or electro-osmosis), (3) apply a temperaturegradient and (4) translate the entire optical trap array through asuspension containing the particles 126 in a manner like a fishing net.Experimentation has determined the particles 134 can be filled into theoptical traps 132 starting with a particle concentration of about 10⁻⁴μm⁻³ and a reasonable flow rate of about 100 μm/sec to fill one row ofthe line 124 or an array pattern in about one minute of time. A fullydeveloped array of the particles 126 can be made permanent bytransferring the array onto a substrate or by gelling the fluid which issuspending the particles. Such a procedure also can allow constructionof a large variety of different particle arrays and coupled arrays ofthe particles 126. Using the previously-described characteristics andfunctionalities of the optical traps 132, each of the particles 126 canalso be further interrogated, imaged and manipulated for operationaluses and investigative purposes.

[0049] In yet another embodiment the optical traps 132 can bedynamically changed responsive to a specific optical requirement, whichcan be effected by use of a computer program with desired instructionalinformation such that one or more of the optical traps 132 can be usedto modify, remove, or add particles at various optical trap sites orallow various manipulations of a single object. Further, one or more ofthe optical traps 132 can be moved and their character changed (such aschanging the shape or strength of the trap) for dynamic manipulation ofany object, such as a cell of a plant or animal. This can beparticularly advantageous when manipulating a delicate structure or whenthere is need to perform complex manipulations of an object. Heretofore,such objects were handles by a single brute force trap which could causedamage to the object or not provide the degrees of freedom often neededto perform a desired function.

[0050] In addition, in another process the particles 126 can bedynamically sorted by size. One can also image an array of the particles126 in the manner shown in FIG. 10. A microscope 138 can image theparticles 126, and a personal computer 140 can identify the particles126 and calculate a phase only hologram 142 (for the diffractive opticalelement 144 of FIG. 8) to trap said particles. A computer controlledspatial light modulator 143 can then implement the computer designedhologram 142 by causing application of a pattern of phase modulations tothe laser beam 144. This can also be dynamically varied for any of avariety of purposes. The modified laser beam 148 (also see the severallaser beams 106, 108 in FIG. 8) are focused by the microscope to createan array of the optical traps 132 (also known as tweezers) which trapsthe particles 126 on image screen 150. Each of the particles 126 canthen be individually manipulated to assemble a desired structure to sortthe particles 126 or to otherwise manipulate, inspect or alter the shapeof the object of interest.

Use of Light in the Visible and UV Spectrum

[0051] In a preferred embodiment of the invention, visible lighttweezers can be used advantageously. In other forms of the invention,for particular sizes of materials matched to ultraviolet light or foruses which are less sensitive to ultraviolet light, the invention canalso be expanded to wavelengths shorter than visible light, includingultraviolet light. Heretofore, tweezers for use in living biologicalmaterial have been formed from infrared light for the reasons describedhereinbefore. Optical tweezers in general can damage biological systemsthrough at least three principal mechanisms: (1) mechanically disruptingphysical interconnections; (2) heating; and (3) in the case ofbiomaterials, photochemical transformation of biomolecules (these aremerely exemplary mechanisms and other mechanisms are possible). Thefirst mechanism includes processes such as drawing into the optical trapthe phospholipids constituting a membrane and then expelling them asmicelles or vesicles. Such processes are inherent in the operation ofoptical tweezers and do not depend on the wavelength of light beingused. These destructive processes can be minimized by using the leastpossible and most efficient trapping force for a given application.Heating results from absorption of the optical trapping photons, astypified by the destruction of hemoglobin-rich red blood cells by greenlaser light. Most biological materials, however, are essentiallytransparent to visible light and in fact absorb more strongly in theinfrared. For example, water has an absorption coefficient of aboutμ_(∂)=3×10⁻⁴ cm⁻¹ at λ=500 nm (visible range) compared with μ_(∂)=0.1cm⁻¹ at λ=1 μm (infrared range). Infrared based optical traps shouldtherefore heat water some 300 times more efficiently than visible lightbased traps. The difference is far less pronounced for other componentsof biological systems. For example, most proteins and polysaccharideshave molar absorption coefficients of: μ_(∂)≈0.1 cm⁻¹ /M for visiblelight and μ_(∂)≈0.01 cm⁻¹/M for infrared radiation. Hemoglobin is anexception, with a comparatively enormous molar absorption coefficient ofμ_(∂)≈10⁴ cm⁻¹/M in the visible light part of the spectrum. In theabsence of such a strong absorption condition, visible light should leadto no worse heating than infrared, and indeed may well be preferablebecause of the prevalence of water in biological systems.

[0052] Photochemical transformations proceed either through resonantabsorption of one or more photons to a discrete molecular state, orthrough non-resonant absorption to a broad molecular band. Most relevantresonant transitions take place in the infrared (for vibrationaltransitions) to the visible (for electronic transitions). Most relevantresonant transitions depend so strongly on the frequency of light,however, that they are highly unlikely to be driven by the monochromaticlight from any particular infrared or visible laser. Transitions tobroad bands take place mostly in the ultraviolet end of the opticalspectrum and so should not be driven either by infrared light or byvisible light.

[0053] “Opticution” (described hereinbefore) is believed to be drivenprincipally by photochemistry, rather than by heating or mechanicaldisruption, and thus it is important to understand why visible (orultraviolet in some cases) optical tweezers can result in deleteriouseffects on biological and other materials having similar photochemistryresponses (or other optically driven events which lead to deleteriouseffects in any type of material, such as light sensitive chemicalstates, light sensitive electronic states or even sensitive mechanicalstructures at the microscopic level). Such materials can include, forexample, small molecule drugs, doped semiconductors, high temperaturesuperconductors, catalysts and low melting point metals.

[0054] For example, living organisms' resistance to photochemicaldegradation at visible wavelengths would appear to be a naturalbyproduct of their evolution in sunlight. However, the flux of visiblelight from the sun is smaller than that in a typical 1 mW opticaltweezer by some six orders of magnitude. The intense illumination at thefocal spot of an optical tweezer greatly increases the rate ofmultiple-photon absorption in which two or more photons cooperate todrive a single optical transition. Multiple photon events requirephotons to arrive simultaneously, and so the level of occurrences ofsuch events depend strongly on the light intensity. The strong focus ofan optical trap does indeed provide the high-intensity light environmentneeded to drive such multiple photon processes.

[0055] Multiphoton absorption can be more damaging in visible tweezersthan in infrared due to the simultaneous absorption of two visiblephotons which delivers the equivalent energy of a single ultravioletphoton. Likewise this can be extended to ultraviolet photons havingwavelengths in which two or more such photons are required to deliverlight energy which would alter the chemical, biological, electronic ormechanical state of a material. Two-photon absorption of infrared light,on the other hand, delivers the equivalent energy of a visible photonand therefore does not usually suffice to drive photochemical or likeoptical transformations. Achieving relevant photochemistry events withinfrared light would therefore require three- or even four-photonabsorption. Because higher-order absorption processes are less likelythan lower order processes, visible optical tweezers appear to be morelikely than infrared tweezers to induce deleterious photochemistry, suchas chromosome recombinations in biological materials. This is believedto be the mechanism by which tightly focused pulses of light at λ=532 nmform an optical scalpel capable of precisely cutting chromosomes.

[0056] The approximate rate W_(n) of an n-photon absorption in the focalvolume of an optical tweezer should scale as follows:${W_{n} \propto \left( {\frac{P}{\hslash \quad c}\frac{\sigma}{\lambda}} \right)^{n}},$

[0057] where P is the power in the beam and σ is the photon capturecross-section for the absorber. As is shown by the above equation,lower-order processes at shorter wavelengths occur much more frequentlythan higher-order processes at longer wavelengths, at least for beams ofequal power.

[0058] Optical tweezers, however, are far more efficient when createdwith light of shorter wavelengths than infrared light (e.g., visible andin some cases ultraviolet). As a result, such optical tweezers requiremuch less power to match the trapping force of infrared tweezers. Thiscomparative advantage of such tweezers opens the door to their use formicromanipulation of biological materials (and for any other highlylight sensitive materials as described hereinbefore). The magnitude ofthe optical gradient force drawing dielectric material to the focus ofan optical trap scales roughly with the inverse fourth power of λ in theRayleigh approximation: ${F \propto \frac{P}{\lambda^{4}}},$

[0059] Therefore a visible trap operating at, for example, λ=532 nmrequires only {fraction (1/16)} the power to achieve the same trappingforce as an infrared trap operating at λ=1064 nm. The relative reductionin power immediately translates into a reduction in the rate W₂ oftwo-photon absorption events for the visible trap. Therefore, thelikelihood of substantial damage is drastically reduced and can evenenable infliction of virtually no damage to the subject material.Furthermore, by careful selection of the wavelength of light used(visible or even ultraviolet in some cases), absorption windows of amaterial being inspected can be used to select the wavelength of lightto reduce the absorption of light and thereby reduce the damage oralteration of the material.

[0060] The trapping efficiency of an optical tweezer can be increasedstill further, and the power requirements correspondingly reduced, byappropriately shaping the wavefront of the trapping beam. For example,it has previously been demonstrated that an optical trap constructedfrom a donut-mode laser beam whose intensity vanishes at the opticalaxis requires far less power to achieve the axial trapping force of aconventional optical tweezer formed with a Gaussian TEM_(∞) mode. Whileit is clear that shaping the wavefront can improve trapping efficiency,thereby reducing two-photon absorption, no studies have reported anoptimal wavefront profile. Therefore, still further improvements areavailable with engineering of the trapping wavefront's characteristics.

[0061] While the time-averaged power establishes the trapping force ofan optical tweezer, its peak power sets the rate of occurrence oftwo-photon processes. Consequently, continuous-wave visible opticaltweezers should also be less damaging than traps derived from pulsedlasers.

[0062] The local irradiation, and thus the rate of two-photon processes,can be reduced still further by applying multiple separate traps to asystem, as described hereinbefore, rather than just one trap.Distributing the trapping force on an extended sample among N opticaltweezers therefore reduces W₂ by a factor of N.

[0063] A system (biological or otherwise) with discrete photosensitivecomponents can be further trapped with visible (or ultraviolet in somecases) light tweezers, provided that care is taken to position the lighttraps away from sensitive areas of the subject material.

[0064] Further, some samples, such as many biological materials, absorblight strongly in the visible range and therefore cannot be trapped withvisible optical tweezers. For the great number of systems largelytransparent to visible light, various steps can be used to minimize therate of deleterious nonlinear optical processes. Without limitation,these can include:

[0065] 1. Given a choice of a range of visible light wavelengths, onecan avoid the use of light wavelengths associated with strongabsorptions characteristic of the type of material.

[0066] 2. One can create traps with a continuous-wave (CW) laser, ratherthan with a pulsed laser.

[0067] 3. One can trap an extended sample at multiple points, ratherthan at just one.

[0068] 4. One can make each trap as efficient as possible. For example,one can take advantage of wavefront shaping to minimize the powerrequired to achieve a desired trapping force.

[0069] The concepts of creating traps with a continuous-wave (CW) laserand avoiding those associated with strong absorptions can be appliedgenerically to all optical tweezer systems. Trapping extended samples atmultiple points and maximizing the efficiency of each trap, however,could be more difficult to implement in conventional optical tweezersystems, but are well within the domain of intended uses of holographicoptical tweezers. In particular, holographic optical tweezers (“HOTs”)can create an arbitrary number N of optical traps in arbitrary positionsso as to trap an extended biological sample (or other highly lightsensitive material) at multiple points. The simplest of these multipletrapping patterns also could be created by rapidly scanning a singletweezer among the desired array of traps. This can be particularlyuseful in biological samples, or other highly light sensitive materials,since not all of the power needs to be imparted on a single point of thesample. By instead distributing the power over a number of points, theoverall damage to the sample will be significantly reduced in a mannersimilar to a “bed of nails.” Achieving a desired time-averaged trappingforce in each of the N scanned traps, however, would require N times thepeak power in each trap. Consequently, HOTs have an inherent advantageover scanned tweezers when it comes to trapping materialsnondestructively.

[0070] HOTs also can produce more complex continuously evolving patternsof traps than can scanned-tweezer systems. This would be an advantage ifoptical tweezer manipulation is intended to move or sort biological orhighly light sensitive materials.

[0071] Holographic optical tweezers also can tailor the wavefronts ofthe individual beams making up the array of traps and can direct eachbeam accurately into the trapping system's focusing optics.Consequently, optical tweezers formed with a HOT system can dynamicallyminimize the amount of power needed to achieve a desired trapping force.

[0072] The above qualitative example guidelines can be used forminimizing the rate of radiation or other light initiated damageinflicted on biological systems (or other highly light sensitivematerials) by optical trapping with visible light. By following theseguidelines, it is possible to obtain acceptable small rate of damage fora particular application. The use of visible light in an HOT systemwould therefore have at least several advantages for applications tobiological systems:

[0073] Optical efficiency. Microscope objective lenses suitable forforming optical tweezers typically are optimized for use at visible andultraviolet wavelengths and suffer from a variety of defects when usedto transmit infrared light. Trapping with visible light thus takesoptimal advantage of the designed properties of conventional optics.Infrared systems, by contrast either must use more costlyspecial-purpose optics, or else will suffer from optical aberrationswhich will somewhat diminish their potential benefits relative tovisible trapping systems.

[0074] Safety. The human visual system includes a protective blinkreflex which reduces the chance that a stray beam of light from avisible trapping system could damage a user's eyesight. No such reflexprotects a user's vision in the infrared.

[0075] Ease of alignment. Visible optical trains are much easier toalign than infrared.

[0076] Availability of two-photon processes. Two-photon processes can beuseful in some biological applications, for instance in creating opticalscissors and scalpels. The same optical train which createsdamage-minimizing configurations of visible optical traps can bereconfigured in real time to produce individual beams optimized fortwo-photon absorption, but with maximum power usage efficiency. Thus thesame system could trap, cut, and generally induce photochemicaltransformations in its samples using a single laser for excitation tofor one or a matrix of optical traps.

[0077] Reduced heating. Infrared systems probably heat their samplesthrough direct absorption by water to a greater extent than do visiblesystems. This excess heating could explain some of the damage reportedin infrared trapping experiments on living systems.

[0078] Improved trapping accuracy. The trapping volume of an opticaltweezer scales with the wavelength of light. Visible light therefore cantrap more accurately localized regions than infrared.

[0079] Improved tracking accuracy. Optical traps sometimes are used totrack the motions of trapped objects, for instance through the timeevolution of the light scattered by trapped particles into the farfield. The resolution of such tracking techniques scales with thewavelength of light and thus would be improved with traps formed invisible light relative to infrared.

[0080] It is also possible that a variety of biological and alsononbiological materials can be manipulated in a manner described abovewith varying wavelengths, laser types, and experimental conditions. Theparticular parameters that are used will be dependent upon the materialto be manipulated and the optical, chemical, mechanical and electricalstates which are sensitive to light. More particularly for example, theabsorption characteristics of the material at certain wavelengths in thevisible range may have a significant bearing on the wavelength of thelaser beam used for the manipulation. For example, the use of certaintypes of green laser light can be successfully used with certainmaterials but not with others (such as chloroplasts in certain types ofplants). For nonbiological materials, such as electronic devices, onecan select visible light wavelengths which do not exhibit strongabsorption by the components of the device.

[0081] It is also noted that the visible portion of the spectrum hasoften been considered to be in the range of about 400 nm to about 700nm. It is possible, however, that a broader wavelength range could beused in accordance with the broader aspects of the invention. Forexample, the window of transparency for water is between about 200 nmand about 800 nm, and an even larger range could be used in certainsituations.

[0082] The following non-limiting examples demonstrate the efficacy ofvisible optical tweezers for manipulating living biological samples. Inparticular, we have demonstrated long-term trapping using light at λ=532nm using a frequency-doubled Nd:YVO₄ laser.

EXAMPLE I

[0083] Large numbers of yeast cells (generic varieties from a package ofFleischman's Yeast) have been trapped in culture medium and haveobserved several generations of budding during continuous illumination.During these tests, light having a wavelength of 532 nm from afrequency-doubled Nd:YV O₄ laser was used to trap the yeast cells. Theyeast included various strains of S. cerevisiae in aqueous solution atroom temperature. Individual cells were trapped with about 1 mW ofcontinuous-wave laser light. In one demonstration, sixteen cells wereconfined to a four by four array. Of these sixteen cells, about halfappeared to be budding daughter cells and forming colonies after sixhours.

EXAMPLE II

[0084] Light having a wavelength of 532 nm from a frequency-doubledNd:YV O₄ laser was used to trap a plurality of cheek epithelial cells.Swabbed cells were suspended in aqueous solution at room temperature anddeposited onto glass cover slips. Optical tweezers were trained on thenuclei and vacuoles of various cells for up to ten minutes. When regionsof the cell membrane were trapped strongly enough to displace asuspended cell through its culture medium, its internal processes didnot appear to be significantly, as determined by visual inspection.Normal cell function appeared to resume after the tweezers wereextinguished.

EXAMPLE III

[0085] Light having a wavelength of 532 nm from a frequency-doubledNd:YV O₄ laser was used to trap wheat chancre cells. The cells wereobtained in solid medium and deposited onto glass cover slips beforeoptical trapping at room temperature. Continuous optical trapping wasnot sufficient to disrupt the cell wall. Visual inspection ofilluminated cells provides qualitative results similar to those obtainedwith cheek epithelial cells.

[0086] While preferred embodiments of the invention have been shown anddescribed, it will be clear to those skilled in the art that variouschanges and modifications can be made without departing from theinvention in its broader aspects as set forth in the claims providedhereinafter.

What is claimed is:
 1. A method of manipulating a biological materialusing a focused beam of laser light, comprising the steps of: providinga focused beam of laser light in the visible wavelength range to formconditions for an optical trap, the laser light having a wavelength suchthat the selected material exhibits a selected absorption coefficient inthe wavelength range of the laser light which permits manipulationwithout substantial damage to the biological material; providing aplurality of the optical traps with the focused beam of laser light, theplurality of optical traps manipulating the biological material; andcontrolling the power level of each of the optical traps, the laserlight wavelength and the selected resulting absorption coefficient toavoid substantial damage to the biological material.
 2. The method ofclaim 1, wherein the focused beam of laser light includes light in thewavelength of about 400 nm to about 700 nm.
 3. The method of claim 1,wherein the beam of light comprises a continuous-wave laser beam.
 4. Themethod of claim 1, further comprising the step of shaping the wavefrontof the focused beam of light such that the power required to achieve apredetermined trapping force is reduced relative to a focused beam oflight that has not been shaped.
 5. The method of claim 4, wherein thebeam of light comprises a donut mode.
 6. The method of claim 1, whereinthe plurality of optical traps arise from a diffractive optical element.7. The method of claim 1, wherein the power level of each of theplurality of optical traps is controlled to avoid altering the geneticcode by a biologically significant amount of the biological material. 8.A method of manipulating a biological material using a focused beam oflaser light, comprising the steps of: providing a continuous-wave,focused beam of laser light having a wavelength such that the materialpossesses a weak absorption coefficient in the wavelength range of thelaser light; providing at least one optical trap for manipulating adiscrete portion of the biological material; and controlling the powerlevel of the at least one optical traps in conjunction with the weakabsorption coefficient to avoid alteration of a substantial portion ofgenetic code of the biological material.
 9. The method of claim 8,wherein a plurality of the optical traps are formed with the focusedbeam of laser light, wherein each of these traps manipulates a discreteportion of the material.
 10. The method of claim 9, further comprisingthe step of shaping the wavefront of the focused beam of light such thatthe trapping efficiency of the at least one optical trap is increasedrelative to not shaping the focused beam of light.
 11. The method ofclaim 10, wherein the focused beam of light comprises a shape calculatedto minimize absorption by the material.
 12. The method of claim 9,wherein each trapping location is not at a biologically sensitivelocation in the biological material.
 13. A system for manipulating abiological material using a focused beam of laser light, comprising: afocused beam of laser light having a wavelength range wherein themaximum wavelength is less than the minimum wavelength of infraredlight, the focused laser light forming an optical trap and having awavelength such that the biological material possesses a weak absorptioncoefficient in the wavelength range of the laser light; and a pluralityof the optical traps for manipulating discrete portions of thebiological material, wherein the power level of each of the opticaltraps is controlled and along with the weak absorption coefficientenables avoiding at least the alteration of the genetic code of abiologically significant amount of the biological material.
 14. Thesystem of claim 13, wherein the focused beam of laser light is selectedfrom the visible and ultraviolet wavelength range.
 15. The system ofclaim 13, wherein the focused beam of laser light comprises acontinuous-wave laser beam.
 16. The system of claim 15, wherein thewavefront of the focused beam of laser light shaped to achieve apredetermined trapping force is reduced relative to a focused beam oflight that has not been shaped.
 17. The system of claim 13, wherein theplurality of optical traps comprise holographic optical traps.
 18. Amethod of manipulating a material using a focused beam of laser light,comprising the steps of: providing a focused beam of laser light in awavelength range less than the wavelength range for infrared light toform conditions for an optical trap; providing a plurality of opticaltraps for manipulating discrete portions of the material; andcontrolling the power level of each of the optical traps but avoidingsubstantial damage to the material.
 19. The method of claim 18, whereinthe focused beam of laser light is selected from the group consisting ofthe visible and ultraviolet wavelength ranges.
 20. The method of claim18, further including the step of using laser light with a wavelength tomatch an absorption window in the material so as to minimize lightabsorption.
 21. A method of manipulating a material using a focused beamof laser light, comprising the steps of: providing a continuous-wave,focused beam of laser light having a wavelength such that the materialpossesses a weak absorption coefficient in the wavelength range of thelaser light; providing at least one optical trap for manipulating thematerial; and controlling the power level of the at least one opticaltraps and also the weak absorption coefficient to avoid substantialalteration of the material.